STUDIES ON INDOOR FUNGI by James Alexander Scott A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy in Mycology, Graduate Department of Botany in the University of Toronto Copyright by James Alexander Scott, 2001 When I heard the learn’d astronomer, When the proofs, the figures, were ranged in columns before me, When I was shown the charts and diagrams, to add, divide, and measure them, When I sitting heard the astronomer where he lectured with much applause in the lecture-room, How soon unaccountable I became tired and sick, Till rising and gliding out I wander’d off by myself, In the mystical moist night-air, and from time to time, Look’d up in perfect silence at the stars. Walt Whitman, “Leaves of Grass”, 1855 STUDIES ON INDOOR FUNGI James Alexander Scott Department of Botany, University of Toronto 2001 ABSTRACT Fungi are among the most common microbiota in the interiors of buildings, including homes. Indoor fungal contaminants, such as dry-rot, have been known since antiquity and are important agents of structural decay, particularly in Europe. The principal agents of indoor fungal contamination in North America today, however, are anamorphic (asexual) fungi mostly belonging to the phyla Ascomycota and Zygomycota, commonly known as “moulds”. Broadloom dust taken from 369 houses in Wallaceburg, Ontario during winter, 1994, was serial dilution plated, yielding approximately 250 fungal taxa, over 90% of which were moulds. The ten most common taxa were: Alternaria alternata, Aureobasidium pullulans, Eurotium herbariorum, Aspergillus versicolor, Penicillium chrysogenum, Cladosporium cladosporioides, P. spinulosum, Cl. sphaerospermum, As. niger and Trichoderma viride. Chi-square association analysis of this mycoflora revealed several ecological groups including phylloplane-, soil-, and xerophilic food- spoilage fungi. Genotypic variation was investigated in two common dust-borne species, Penicillium brevicompactum and P. chrysogenum. Nine multilocus haplotypes comprising 75 isolates of P. brevicompactum from 50 houses were detected by heteroduplex mobility assay (HMA) of ii polymorphic regions in beta-tubulin (benA), nuclear ribosomal RNA spanning the internal transcribed spacer regions (ITS1-2) and histone 4 (his4) genes. Sequence analysis of the benA and rDNA loci showed two genetically divergent groups. Authentic strains of P. brevicompactum and P. stoloniferum clustered together in the predominant clade, accounting for 86% of isolates. The second lineage contained 14% of isolates, and included collections from the rotting fruit bodies of macrofungi. Similarly, 5 multilocus haplotypes based on acetyl coenzyme-A synthase (acuA), benA, ITS1-2 and thioredoxin reductase (trxB) genes comprised 198 isolates of P. chrysogenum obtained from 109 houses. A strictly clonal pattern of inheritance was observed, indicating the absence of recombination. Phylogenetic analyses of allele sequences segregated the population into three divergent lineages, encompassing 90%, 7% and 3% of the house dust isolates, respectively. Type isolates of P. chrysogenum and its synonym P. notatum clustered within the secondary lineage, confirming this synonymy. No isolates of nomenclatural status clustered within the predominant lineage; however, this clade contained Alexander Fleming’s historically noteworthy penicillin-producing strain from 1929. Similarly, there was no available name for the minor lineage. iii ACKNOWLEDGEMENTS I am profoundly grateful to my advisors, Dave Malloch and Neil Straus, for their thoughtful mentorship, both intentional and unintentional, on all matters of science and life. They have given me gifts of their patience and wisdom that I cannot repay. I thank the members of my supervisory committee, Jim Anderson, David Miller and Richard Summerbell, for their gentle encouragement and sincere enthusiasm which helped me to stay on-track. Linda Kohn and Keith Seifert are thanked for their excellent and thoughful feedback on the thesis in conjunction with the senate oral examination. I could not have completed this thesis nor the research it presents without the friendship, dedication and benevolence of Brenda Koster, Bess Wong and Wendy Untereiner. I have laughed, cried and grown immeasurably from my friendships and discussions, often over beer, pizza or #49, with my fellow graduate students Jacquie Bede, Cameron Currie, Laurie Ketch and Simona Margaritescu, along with many others. I am grateful for the assistance of Len Hutchison, Brett Couch, Wendy Malloch, Colleen McGee, Emily Taylor and Megan Weibe during the early stages of this work. More recently, Michael Warnock helped with proofreading and correcting this thesis. Barry Neville and Shanelle Lum kindly kept me informed of many news items on indoor fungi that otherwise I would certainly have missed. Carolyn Babcock and Steve Peterson graciously provided cultures. I thank John Pogacar for insightful conversations on building science and for help in final thesis preparation. Financial support was provided by NSERC as operating grants and a strategic grant to DM and NS, and a doctoral scholarship to JS. Jim Gloer generously funded my early mycological training. iv Wieland Meyer has been a superlative collaborator on the aspects of this project involving DNA fingerprinting of Penicillium chrysogenum. Steve Peterson, John Pitt and Rob Samson are thanked for many insightful discussions on Penicillium taxonomy. I owe the greatest debt of gratitude to my parents, Kay and Alex Scott, for nurturing my ecclectic interests from a very young age, for their compassion and understanding of the many turns my life has taken, and for the unconditional freedom, support and love that they have given me as I have pursued my dreams. For all this and much more, I dedicate this thesis to them. On a muggy, summer day when I was a very young boy, my cousin, Jim Guillet inspired me to study biology. As we stood together in my grandmother’s garden, Jim explained that the stems of the rhubarb plant could be eaten but that the leaves could not, because they were poisonous. How could it be so? And why? I stared in utter disbelief and hotly challenged this absurd idea, while my parents, grimacing, looked on. I stopped just short of biting into a leaf myself to see its effect. In the very many intervening years since, I have reflected on our exchange countless times. Jim, perhaps unintentionally, taught me four very valuable lessons that day: 1) Nature is fascinating and intricate, her properties and processes are rarely apparent or intuitive; 2) Never be afraid to question any notion proffered as fact, no matter how high the authority; 3) Science embodies a set of methods that can provide insight into the delicate inner workings of Nature when applied thoughtfully and skillfully; and above all, 4) Don’t eat rhubarb leaves. v TABLE OF CONTENTS PAGE ABSTRACT ................................................................................................................ II ACKNOWLEDGEMENTS ....................................................................................................... IV TABLE OF CONTENTS.......................................................................................................... VI LIST OF TABLES ................................................................................................................ X LIST OF FIGURES ............................................................................................................... XI LIST OF APPENDICES.........................................................................................................XIII LIST OF ABBREVIATED TERMS........................................................................................... XIV LIST OF NOMENCLATURAL ABBREVIATIONS ..................................................................... XVI CHAPTER 1. INTRODUCTION .......................................................................................1 The biology of house dust.....................................................................................................................1 Interactions between mites and fungi.............................................................................................3 Fungi in household dust....................................................................................................................3 Indoor sources of dust mycoflora ...................................................................................................5 Penicillium in indoor environments...................................................................................................6 Health effects of exposure to indoor fungi ........................................................................................9 Allergic rhinitis and sinusitis...........................................................................................................10 Type I allergic syndromes...........................................................................................................10 Dust mites and allergy.................................................................................................................11 Hypersensitivity syndromes............................................................................................................12 Asthma...............................................................................................................................................12 Mycotoxins........................................................................................................................................15